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An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens

假单胞菌侵染被荧光葡萄孢菌预处理过的葡萄叶片实验

CG Charlotte Gruau
Patricia Trotel-Aziz Patricia Trotel-Aziz
BV Bas Verhagen
SV Sandra Villaume
Fanja Rabenoelina Fanja Rabenoelina
BC Barbara Courteaux
Christophe Clément Christophe Clément
Fabienne Baillieul Fabienne Baillieul
Aziz Aziz Aziz Aziz

发布: 2016年10月05日第6卷第19期 DOI: 10.21769/BioProtoc.1943 浏览次数: 13114

评审: Arsalan DaudiRupesh PaudyalAnonymous reviewer(s)

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The authors used this protocol in:

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Abstract

Grapevine (Vitis vinifera L.) is susceptible to an array of diseases among them the grey mold caused by the necrotrophic fungus Botrytis cinerea that decreases grape productivity and quality. To ensure a satisfactory yield and harvest quality numerous chemical fungicides are required, but they have serious drawbacks. One alternative is the use of beneficial bacteria to improve plant health. Pseudomonas fluorescens has been shown to trigger a plant-mediated resistance response in aboveground plant tissues against fungal, oomycete, bacterial, and viral pathogens. Triggered plant resistance exploits mechanisms of the plant immune system through a priming state that provides plants with enhanced capacity for rapid and strong activation of defense responses after pathogen infection, resulting in a lower fitness-cost. The primed responses by beneficial bacteria include induced expression of defense-related genes, cell wall reinforcement, and the production of secondary metabolites after pathogen infection. In this protocol, we describe the experimental design to evaluate the priming state of grapevine plants by the beneficial bacterium Pseudomonas fluorescens PTA-CT2 and their resistance level to Botrytis cinerea according to Verhagen et al. (2011) and Gruau et al. (2015).

Keywords: Biocontrol (生物防治) search Pseudomonas (铜绿假单胞菌) search Grapevine (小道消息) search Priming (启动) search Botrytis (灰) search

Materials and Reagents

  1. 25 mm culture tubes (VWR, catalog number: 212-0304 )
  2. Conical tubes (50 ml) (Corning, Falcon®, catalog number: 352070 )
  3. Sterile spatula (VWR, catalog number: 612-1561 )
  4. Falcon® cell strainer 100 µm nylon (Corning, Falcon®, catalog number: 352360 )
  5. 1.5 ml microtubes (Treff, catalog number: 96.07246.9.01 )
  6. 2 ml microtubes (Treff, catalog number: 96.09329.9.01 )
  7. Whatman paper ashless discs 90 mm diameter (Sigma-Aldrich, catalog number: WHA1441090 )
  8. Petri dishes (90 x 14 mm) (VWR, catalog number: 391-0439 )
  9. Grapevine (Vitis vinifera cv. Chardonnay 7535) plants obtained by micropropagation from nodal explants on agar Murashige & Skoog (MS) medium (Aziz et al., 2006) in 25 mm culture tubes
  10. Pseudomonas fluorescens PTA-CT2 (Trotel-Aziz et al., 2008)
  11. Botrytis cinerea conidia (Bc 630) isolated from infected grape at ripening by INRA Versailles, France
  12. Distilled water from Aquadem blue range (Véolia Water Aquadem)
  13. Distilled water sterilized with autoclave Avor Getinge
  14. Ethanol (from Alcool absolu) (Charbonneaux Brabant, catalog number: 3077319001001 )
  15. Murashige & Skoog medium (MS) (Duchefa Biochimie, catalog number: M0231.0001 )
  16. Glycerol (VWR, catalog number: 24387-292 )
  17. Luria Bertani liquid medium (LB) (Duchefa Biochimie, catalog number: L1703.0500 )
  18. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M2773 )
  19. BactoTM agar (BD, catalog number: 214010 )
  20. Agar tomato medium (see Recipes)

Equipment

  1. Plant growth chamber with photoperiod (SANYO, model: MLR-351 H )
  2. Horizontal laminar flow cabinet (ASTEC Microflow, model: Horizontal laminar flow workstation )
  3. Long sterile metal clamp (VWR, catalog number: 232-0084 )
  4. Magenta boxes (Sigma-Aldrich, catalog number: V8505 )
  5. Microbiological safety cabinet (Angelantoni Life Science, model: VBH Compact )
  6. Sterile pipettes (VWR, catalog number: VHPA23004 )
  7. 28 °C incubator shaker (New Brunswick Scientific, model: C24 )
  8. Variable speed refrigerated centrifuge with rotor having number of rotor cavities x nominal largest load (capacity: 8 x 50 ml) (Eppendorf, model: 5804R )
  9. Spectrophotometer (Bio-Rad Laboratories, model: SmartSpec 3000 )
  10. Vortex mixer (Vortex Genie 2 untimed mixer) (VWR, catalog number: 58815-232 )
  11. Malassez counting chamber (Marienfeld, catalog number: 0640610 )
  12. Glass atomizer (VWR, catalog number: 552-0031 )
  13. Camera (Nikon, model: D7000 )

Software

  1. APS Assess 2.0 software (APS, Item No: 43696M5, ISBN 978-0-89054-369-6)

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Gruau, C., Trotel-Aziz, P., Verhagen, B., Villaume, S., Rabenoelina, F., Courteaux, B., Clément, C., Baillieul, F. and Aziz, A. (2016). An Assay to Study Botrytis cinerea-infected Grapevine Leaves Primed with Pseudomonas fluorescens . Bio-protocol 6(19): e1943. DOI: 10.21769/BioProtoc.1943.

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分类

植物科学 > 植物免疫 > 病害生物测定

植物科学 > 植物免疫 > 信号感知与传递

微生物学 > 微生物-宿主相互作用 > 体内实验模型 > 植物

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